Field of the Invention
The present invention relates to an instrument and a method which relate to separation and analysis of membrane vesicles in the biological field, the biochemical field, the biotechnological field, the medical field, and the medicinal field.
Description of the Related Art
In the related art, in a structure such as a membrane vesicle from a living body such as a cell or a cell organelle or an artificial membrane vesicle covered by a lipid bilayer membrane, the content of this structure or a substance held on the lipid bilayer membrane has been analyzed.
In recent years, as a method for transmitting information between cells, a method using an exosome which is a vesicle having a lipid bilayer membrane has attracted attention.
Exosomes contain protein, mRNA, micro RNA (miRNA), DNA, or the like therein, and are membrane vesicles known to be able to transmit information to a destination cell by moving between cells. For example, it is known that in cells in which exosomes containing micro RNA from cancer cells are present, an immune function is activated or metastasis ability is acquired.
In exosomes, in addition to genetic information and other signal factors within cells from which exosomes are released, factors that control functions of other cells to which the exosomes are accepted are included. Therefore, it is considered that it is possible to utilize exosomes as a new biomarker source for diagnosing diseases.
For example, Japanese Unexamined Patent Application, First Publication No. 2013-102768 and Published Japanese Translation No. 2010-534480 of the PCT International Publication disclose that cancer or an adverse pregnancy outcome can be diagnosed by analyzing miRNA within an exosome.
Published Japanese Translation No. 2011-524164 of the PCT International Publication discloses that each RNA is measured for determining the efficiency of treatment using a small interfering RNA (siRNA) therapeutic agent or a miRNA therapeutic agent.
Published Japanese Translation No. 2013-516619 of the PCT International Publication discloses that a protein marker which becomes an indicator of risk of onset of a cardiovascular event is detected.
Published Japanese Translation No. 2010-517048 of the PCT International Publication discloses that a disease such as cancer or infertility associated with the production of an autoantibody can be diagnosed by measuring the level of immunoreactive autoantibody.
Japanese Unexamined Patent Application, First Publication No. 2013-7698 discloses that a vesicle stress response and a renal disease which is associated with the response can be detected by measuring aquaporin 1 of an exosome in urine.
An exosome can be prepared after being separated from a sample which can contain an exosome through separation such as ultracentrifugation or density gradient ultracentrifugation using a density difference. In addition, an exosome can also be separated through methods disclosed in Published Japanese Translation No. 2003-531864 of the PCT International Publication or Published Japanese Translation No. 2002-535665 of the PCT International Publication. In addition, a kit for separating and purifying an exosome is commercially available (for example, ExoQuick of System Biosciences, Inc. or Exosome Isolation of Life Technologies).
For example, in the case of separating an exosome through centrifugation, it is impossible to avoid impurities other than the exosome from being mixed in the separated exosome, and therefore, there is a limit to improve analysis accuracy and reproducibility.
In addition, in the ultracentrifugation, the density gradient ultracentrifugation, or the like, the work procedure for separating an exosome is complicated and it requires a long period of time to separate and purify an exosome.
In addition, a separation kit in the related art which simply separates an exosome has inferior reliability since purification of an exosome is insufficiently performed.
It is also known that an exosome is selectively separated and purified using tetraspanin existing in a lipid bilayer membrane of an exosome. Tetraspanin is, for example, a 4-pass transmembrane type membrane protein family known to have 33 members in a human. Particularly, CD9, CD63, and CD81 are known as exosome markers. However, in the case where an exosome is separated and purified using an anti-tetraspanin monoclonal antibody, in some cases, an ununiformity is caused in an exosome finally separated and purified depending on the type of antigen to be separated.
In addition, in some cases, the membrane structure of an exosome is destroyed in order to analyze the exosome. In this case, the content of the exosome is diluted in the exosome destruction process. Even if it is necessary to concentrate a product obtained by destroying an exosome in a diluted state, it is difficult to concentrate the product since there may be components which are degenerated or lost in the concentration process. In addition, in the case where protein of an exosome is to be analyzed, it is impossible to amplify protein to be analyzed in vitro unlike in the case of a nucleic acid. Therefore, it is necessary to analyze an exosome without diluting the exosome, if possible.
The present invention has been made in consideration of the above-described circumstances, and an object of the present invention is to provide a membrane vesicle recovery device in which a membrane vesicle is not destroyed and constituents of the membrane vesicle are barely diluted, and which can selectively recover a membrane vesicle; a membrane vesicle recovery method; and a membrane vesicle analysis method which is simple and is excellent in accuracy and reproducibility.